Investigation of the role of autophagy in the resistance of pancreatic cancer to radiation therapy. Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the U.S. with a dismal 5-year survival rate of 3-5%. A hallmark of this aggressive cancer is its resistance to existing therapies including chemotherapy and radiation. We explored whether a component of this resistance was attributable to altered cell survival/death pathways. During these studies we made the novel finding that virtually all human PDAC primary tumors and cell lines have elevated basal autophagy. We found that inhibition of autophagy in PDAC by pharmacological or genetic means causes pronounced elevation of reactive oxygen species (ROS) and increased DNA double strand breaks, leading to marked growth arrest in vitro as well as tumor regression in vivo. Thus, autophagy is required for PDAC growth by preventing accumulation of high levels of DNA damage. Importantly, the DNA damage caused by autophagy inhibition is synergistic with radiation. We therefore hypothesize that autophagy has a critical role in the resistance of PDAC to chemotherapy and radiation in the clinical setting. Since potent autophagy inhibitors, such as chloroquine (CQ) and hydroxychloroquine (HCQ), are available and FDA approved, this work has immediate clinical application to the treatment of PDAC and other tumor types with elevated autophagy. As resistance to radiotherapy with subsequent local failure is a significant cause of morbidity and mortality in PDAC patients, these studies have the potential for a transformative impact. Based on this, we propose to 1) define molecular predictors for response to autophagy inhibition as a radiosensitizer in PDAC cell lines and xenografts, 2) establish the role of autophagy in the histopathogenesis primary PDAC and in sensitivity of native tumors to radiation and chemotherapy using genetically engineered mouse models, and 3) to assess the potential of autophagy inhibition as a radiosensitizer in tumors from human PDAC patients treated with radiation and the autophagy inhibitor hydroxychloroquine. Aim 1 will use a collection of >70 mouse and human PDAC lines to determine if various genotypes (combinations of KRAS, p53, Ink4a, Smad4, Lkb1, and PTEN mutations), global gene copy number and mRNA expression profiles can predict response to HCQ as a radiosensitizer. Aim 2 will employ validated mouse models of PDAC progression, autophagy reporters, and pharmacologic and genetic approaches to inactivate autophagy in evolving tumors in vivo elucidate the contribution of autophagy to the development and radioresistance of tumors arising in their native setting. Aim 3 will utilize tumors from patients enrolled on a clinical trial assessing the efficacy of the autophagy inhibitor HCQ as a radiosensitizer in PDAC to validate in vivo autophagy inhibition in tumors, assess histologic response, and validate molecular predictors from Aim 1 in patient samples. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is highly lethal and current treatments such as chemotherapy and radiation are only minimally effective due to its profound therapeutic resistance. This proposal seeks to build on data from our laboratory showing that a process called autophagy may be important in the resistance of these tumors to radiation therapy.